Sperm cell processing and preservation systems

ABSTRACT

Semen and sperm cell processing and preservation systems, and methods of producing a mammal and methods of producing mammalian embryos are disclosed. The present invention is directed to sperm cell preservation, fertilization, and insemination, maintaining or enhancing sperm quality and addressing one or more sperm cell characteristics, such as viability, motility, functionality, fertilization rates, and pregnancy rates. Further, sperm cell characteristics may be addressed within the context of various collection, handling, separation, storage, transportation, usage, fertilization, or insemination techniques.

This application is a continuation application of prior U.S. patentapplication Ser. No. 10/340,881, filed Jan. 9, 2003, which claims thebenefit of prior U.S. Provisional Application No. 60/410,884, filed Sep.13, 2002, each said foregoing application and any priority case herebyincorporated herein by reference.

BACKGROUND OF THE INVENTION

Fertilization techniques such as artificial insemination and in vitrofertilization techniques have been conducted in attempt to increase thefertility rates of livestock. Furthermore, effective pre-selection ofsex has been accomplished in many species of livestock following thedevelopment of safe and reliable methods of sorting, generally, orspecifically separating sperm cells into enriched X chromosome bearingand Y chromosome bearing populations. Separation of X chromosome bearingsperm cells from Y chromosome bearing sperm cells, as well ascollection, handling, sorting, separation, storage, transportation, use,fertilization, or insemination techniques, or sperm cell and semenprocessing generally, can be accomplished as disclosed herein and asdisclosed by various patent applications, for example: PCT/US99/17165;PCT/US01/45023; PCT/US01/15150; PCT/US98/27909; PCT/US01/45237,PCT/US01/18879, PCT/US/00/30155, PCT/US01/02304, U.S. Pat. No.6,071,689, U.S. Pat. No. 6,372,422, U.S. divisional application Ser. No.10/081,955, U.S. provisional application No. 60/400,486, and U.S.provisional application No. 60/400,971, each included in Exhibit Aattached, and each hereby incorporated by reference herein.

Although the various devices and methods of sperm cell processing,generally, and the collection, handling, separation, storage,transportation, usage, fertilization, and insemination of sperm cellshave been improved over the past several years, significant problemsremain with respect to maintaining sperm quality, such as viability,motility, functionality, and preservation and stimulation relative tosuch techniques, especially with regard to artificial insemination (invivo) and in vitro fertilization (IVF) procedures. One potentialconsequence is the reduction in fertility rates. Sperm quality, such asthe viability of sperm separated into enriched X-chromosome bearing andY-chromosome bearing populations could be compared directly in-vitro(for example, in conjunction with IVF procedures) and in-vivo (forexample, in conjunction with artificial insemination procedures), isgenerally reduced during traditional sperm cell processing. Moregenerally, traditional processing techniques addressing sperm viability,motility, functionality, preservation, stimulation, fertilization, andinsemination may have not yielded preferred fertility rates,insemination rates, fertilization rates, or sperm quality, generally.

As one example of traditional sperm processing, techniques of spermsorting for the breeding of mammals may be limited, for example, withregard to sperm cell quality, such as sperm cell viability, andfertility rates, in circumstances wherein the sorter apparatus is arelatively long distance from the males or when the sorter apparatus isa relatively long distance from the receiving females to be inseminatedor otherwise fertilized with processed sperm. Some of the techniquesincorporated by reference may achieve to some degree sperm cellviability, motility, or functionality, and desirable fertility rates,while addressing, for example, sperm preservation during transportationto the sorter apparatus. However, further techniques achievingsufficient sperm cell quality, such as viability, motility,functionality, stimulation, and preservation, and maintained or enhancedfertility rates, insemination rates, fertilization rates, may bedesirable, especially for any one or a combination of various spermprocessing steps, such as, for example, collection, handling,separation, storage, transportation, usage, fertilization, orinsemination of semen or sperm cells, especially the preservation of thesorted sperm cells from the sorter apparatus to the site offertilization (potentially via collected individual samples or“straws”), or at any other stage of sperm processing.

As another example of the limitations of traditional sperm processing,traditional processing techniques may be limited with regard to spermcell quality, generally, such as sperm cell viability, motility,functionality, stimulation, and preservation, as well as fertilityrates, insemination rates, fertilization rates, due to the type and theextent processing involved. Known processing techniques such aspreservation or sorting, for example, may degrade sperm quality, andfurther reduce fertility rates, insemination rates, and fertilizationrates. The degradation of sperm quality and/or the reduction offertility rates, insemination rates, and fertilization rates may havepreviously proven problematic in circumstances that may have requiredfurther steps of sperm cell preservation, such as in circumstanceswherein the sorter apparatus is a relatively long distance from themales or when the sorter apparatus is a relatively long distance fromthe receiving females to be inseminated or otherwise fertilized withprocessed sperm.

It may have been traditionally understood that additional processingsteps in the processing of sperm cells or semen having a concentrationof sperm cells would degrade sperm quality and reduce fertility rates,insemination rates, and fertilization rates to such an extent thatadditional processing steps, generally, would be avoided. Specifically,it may also have been traditionally understood that processing stepssuch as preservation, specifically cryopreservation, and sorting mightbe too damaging to the sperm cells, especially in processing sequencesincorporating both cryopreservation and sorting. Furthermore, it mayhave even been suggested and taught in traditional methods thatpreservation of sperm, such as cryopreservation, provided after previousprocessing steps so as to preserve the sperm for later procedures, suchas for in vivo or in vitro techniques, could not be accomplished withoutcompromising the sperm cells and the technique itself to such a degreethat the results of the later procedures would be detrimentallyaffected. Accordingly, such concerns may have even been demonstrated intraditional sperm cell processing procedures, teaching away fromsubsequent processing steps such as sorting and cryopreservation, orprocessing steps incorporating multiple cryopreservation steps.

SUMMARY OF THE INVENTION

The present invention addresses the variety of previously identified andpotentially unaddressed problems associated with reduced sperm cellquality, such as viability, motility, functionality, stimulation, and ofpreservation. Sperm cells that may have been or will be processed by oneor more steps of collection, handling, separation, storage,transportation, usage, fertilization, or insemination and potentialreductions in fertility rates, insemination rates, or fertilizationrates are further addressed. The instant invention also addresses thevariety of problems associated with sperm cell quality of sperm cellsthat have been separated into enriched X-chromosome bearing andY-chromosome bearing populations, potentially sperm cells separated orsorted through techniques such as flow sorting, and the potentialreduction in fertility rates. More generally, the present inventionaddresses traditionally low fertility rates of in vivo artificialinsemination and in vitro fertilization, and sperm cell quality issuessuch as sperm cell viability, motility, functionality, stimulation, andpreservation, as well as fertility rates, insemination rates,fertilization rates, achieving results that may have been unexpected tothose skilled in the art. Additionally, the present invention addressesnumbers of processed sperm potentially needed to obtain desiredpregnancy rates, and further in consideration of pregnancy ratesobtained respective of sorted and unsorted preserved sperm.

Accordingly, broad objects of the present invention are to providesystems to address sperm cell quality, such as viability, motility,functionality, and preservation, and to address fertilization rates,insemination rates, and fertilization rates. Each of the broad objectsof the present invention may be directed to one or more of the variousand previously described concerns.

One object of the present invention is to provide methods and systems ofsemen and sperm cell processing and preservation, and methods andsystems of producing a mammal and methods of producing mammalianembryos. A further object of the present invention is to providepreservation, stimulation, fertilization, and insemination, potentiallyprovided alone or in combination with any one or a combination ofvarious sperm processing steps, such as, for example, collection,handling, separation, storage, transportation, usage, fertilization, orinsemination of semen or sperm cells. One related object is to providesuch systems in relation to the preservation of sperm cells and one ormore sequences of sperm cell processing, such as the preservation ofsorted sperm cells, and further in some instances, for example,preservation to a sorter apparatus, preservation from a sorterapparatus, preservation to the site of fertilization (potentially viaindividual sperm samples or “straws”), or at any other stage of spermprocessing.

A related object of the invention is to provide systems of collection,handling, separation, storage, transportation, usage, fertilization, orinsemination for semen or sperm cells to achieve desirable levels offertility rates. One related goal is to provide systems of collection,handling, separation, storage, transportation, usage, fertilization, orinsemination to maintain and enhance sperm viability, motility,functionality, preservation, stimulation, and levels of fertility rates.

A third object is to address sperm cell quality, such as sperm cellviability, motility, functionality, stimulation, and preservation, aswell as fertility rates, in the context of fertilization andinsemination, and in some embodiments, in the context of in vivo or invitro techniques. A related goal of the present invention is to providesystems that achieve desirable levels of fertility rates. A second goalof the present invention is to provide systems that achieve desirablelevels of fertility rates in combination with collection, handling,separation, storage, transportation, fertilization, or inseminationtechniques, and combinations of such techniques. A third goal of thepresent invention is to provide systems that achieve individual spermsamples of processed sperm cells, such as straws, of desired viability,motility, functionality, preservation, stimulation, or othercharacteristics, or combinations of characteristics, potentiallyresulting in desirable levels of fertility rates. Another related goalis to provide systems that achieve sufficient numbers of processed spermpotentially needed to obtain desired pregnancy rates, and further inconsideration of pregnancy rates obtained respective of processingtechniques, such as the sorting of sperm cells, and in some cases, spermor sperm cells previously preserved.

A further object is to address sperm cell quality, such as sperm cellviability, motility, functionality, stimulation, and preservation, aswell as fertility rates, insemination rates, fertilization rates, forsemen or sperm cells obtained from various species of mammals,including, but not limited to equids, bovids, felids, ovids, canids,buffalo, oxen, elk, or porcine; or obtained from prize, endangered, orrare individuals of a mammal species; or obtained from zoologicalspecimens. A related goal of the present invention, therefore, is toaddress sperm cell quality, generally, such as sperm cell viability,motility, functionality, stimulation, and preservation, as well asfertility rates, insemination rates, fertilization rates, for semen orsperm cells obtained from various species, individuals, and specimens soas to maintain or enhance sperm viability, motility, functionality,preservation, stimulation, fertility rates, or other characteristics, orcombinations of characteristics.

Another object of the invention is to provide systems of collection,handling, separation, storage, transportation, usage, fertilization, orinsemination for semen or sperm cells obtained from various species ofmammals, including, but not limited to equids, bovids, felids, ovids,canids, buffalo, oxen, elk, or porcine; or obtained from prize,endangered, or rare individuals of a mammal species; or obtained fromzoological specimens. A related goal of the present invention,therefore, is to provide systems of collection, handling, separation,storage, transportation, usage, fertilization, or insemination for semenor sperm cells obtained from various species, individuals, and specimensso as to maintain or enhance sperm viability, motility, functionality,preservation, stimulation, fertility rates, or other characteristics, orcombinations of characteristics.

An object of the present invention is to provide systems that achieveindividual samples of processed sperm, such as straws, of desired spermcell quality, such as sperm cell viability, motility, functionality,stimulation, and preservation, or other characteristics, or combinationsof characteristics, as well as providing desirable levels of fertilityrates, insemination rates, or fertilization rates.

Another significant object of the invention is to provide systems ofsperm cell separation that can maintain or enhance a greater sperm cellquality, such as sperm cell viability, motility, functionality,stimulation, and preservation, or other characteristics, or combinationof characteristics, as well as fertility rates, insemination rates,fertilization rates, for sperm cells throughout a flow sorting processsuch as a process incorporating a flow cytometer.

Another significant object of the invention can be to provide systems ofmaintaining or enhancing sperm cells at greater sperm cell quality, suchas sperm cell viability, motility, functionality, stimulation, andpreservation, or other characteristics, or combination ofcharacteristics, as well as fertility rates, insemination rates, orfertilization rates, for purposes of in vivo insemination or in vitrofertilization of various species, such as those described above, or eveninsemination with a low or reduced number of sperm cells compared to theusual number or typical number of sperm cells used in such inseminationprocedures whether or not such sperm cells are separated into enriched Xchromosome bearing or Y chromosome bearing sperm cells.

Naturally, further significant objects and goals of the invention aredisclosed and clarified in the proceeding description of the invention.

Accordingly, to achieve the various objects and goals of the invention,the present invention in one embodiment is a method of sperm cellprocessing, and the steps provided as obtaining sperm cells,cryopreserving the sperm cells, thawing the sperm cells, processing thesperm cells; and cryopreserving the sorted sperm cells. A furtherembodiment of the invention is a method of producing a mammal,comprising the method steps of obtaining sperm cells, cryopreserving thesperm cells, thawing the sperm cells, sorting the sperm cells,cryopreserving the sorted sperm cells, thawing the sorted cryopreservedsperm cells, inseminating at least one egg with the sorted cryopreservedsperm cells, fertilizing the at least one egg, and producing a mammalfrom the at least one fertilized egg.

Furthermore, to achieve the various objects and goals of the invention,the present invention is directed to embodiments providing forartificial insemination (in vivo) and in vitro fertilization procedures.Additionally embodiments may be further directed to the production ofmammals or mammalian embryo, and may further be directed to establishingsperm samples such as straws, pellets, or the like, and the processingof semen.

Additional embodiments of the invention are disclosed throughout thedescription of the invention and in the following claims.

BRIEF DESCRIPTION OF THE DRAWINGS

Each Figure contained in each of the applications set out in Exhibit Aare to be considered hereby incorporated by reference as part of thisdescription of the instant invention.

FIG. 1 is a flow diagram for one embodiment of the present invention.

FIG. 2 is a flow diagram for a second embodiment of the presentinvention, potentially provided in combination with the embodiment ofFIG. 1, in some embodiments.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention discloses semen and sperm cell processing andpreservation techniques and systems of preservation, stimulation,fertilization, and insemination, addressing one or more sperm cellcharacteristics or sperm quality, such as sperm cell viability,motility, functionality, stimulation, and preservation, as well asfertility rates, insemination rates, fertilization rates. Further, spermcell characteristics or sperm quality may be addressed within thecontext of various processing techniques, such as collection, handling,separation, storage, transportation, usage, fertilization, orinsemination techniques.

Sperm cell quality may refer to any one or a combination of the variousattributes of sperm cells previously mentioned or further mentionedherein, such as, for example, viability, motility, functionality,stimulation, and preservation of the sperm, or fertility rates,insemination rates, or fertilization rates corresponding to the sperm(such as in the fertility of the sperm). Sperm cell characteristic mayrefer to any one or a combination of various biological, chemical,physical, physiological, or functional attributes of one or more spermcells, such as chromosome bearing attributes of the cell, or in someembodiments may refer to sperm cell quality as previously described.

Semen or sperm cell processing techniques may refer to any one or acombination of preservation, stimulation, insemination, fertilization,sorting, selection, separation, or thawing, and may be specificallydirected to any one or a combination of collection, handling, selection,storage, transportation, usage, fertilization, or inseminationtechniques.

Sperm samples may refer to a volume containing sperm cells, potentiallyincluding semen, carrier fluid or other materials, and may comprise apellets, straws, or other known forms of sperm samples.

Maintaining or Enhancing the Sperm Quality

Sperm cells may be maintained or enhanced in accordance with the presentinvention, and in some embodiments through sperm processing, such asthrough sperm cell sorting and preservation, and in accordance withfertilization and insemination techniques. Embodiments may providepreservation of the sperm or sperm cells, or other techniques as may bedisclosed in the previously mentioned patent applications and thereferences listed in the List of References to be Incorporated byReference, each application and reference expressly incorporated byreference to the extent consistent with the present description, and asfurther described below.

Sperm cells may also be maintained or enhanced in some embodiments bypreservation and stimulation techniques as further described below, andalso in further combination with the processing, stimulation,preservation, fertilization, and insemination techniques in thepreviously mentioned patent applications and the references listed inthe List of References to be Incorporated by Reference.

Accordingly, the present invention may provide a method of sperm cellprocessing. FIG. 1 shows one process for sperm cell processing inaccordance with the present invention. As shown, the sperm cells may beobtained 10 from a mammalian source, either directly or in variouscombinations of steps, such as through a storage facility. The spermcells may be cryoperserved 20, as further described below. Subsequently,the sperm cells may be thawed 30. Further processing 40, furtherdescribed below, may then be performed on the sperm cells prior toanother cryopreservation of the sperm cells.

Sperm cell processing, generally, and the process previously describedmay be conducted for insemination and fertilization. Therefore, anembodiment of the present invention may provide a method of producing amammal, as shown in FIG. 2. After proceeding with previous processing,or in some embodiments as proceeding with the features 10, 20, 30, 40,and 50, the insemination of the mammal may performed. In one embodiment,the cryopreserved sperm cells may be thawed 60 and the insemination 70and fertilization 80 of at least one egg with the sperm cells may beconducted. A mammal may then be produced 90 from the egg fertilized bythe cryopreserved, processed, cryopreserved, and thawed sperm cells.

Furthermore, embodiments further support in vivo and in vitrotechniques. Accordingly, a female of the species of the mammal fromwhich sperm was obtained may be inseminated with the previouslyprocessed sperm cells, such as sorted and cryopreserved sperm cells aspreviously described. At least one egg of the female may then befertilized and a mammal produced from the at least one fertilized egg.In accordance with in vitro procedures, after insemination andfertilization of at least one egg, a mammalian embryo may be produced,potentially developing into a mammal.

Each of the embodiments previously described may be considereddepartures from traditional sperm cell processing and the production ofmammals and mammalian embryo. It has been traditionally viewed that suchprocessing of sperm cells could not sufficiently ensure sperm quality orprovide adequate rates of fertility, insemination, fertilization, orpregnancy. However, and as been taught in the various references citedherein and incorporated by reference, sperm cells may in fact beprocessed to achieve, for example, the sorting of sperm cells,potentially to differentiate sperm cells as either X chromosome bearingor Y chromosome bearing sperm cells, in some instances to provide forpreselection of the sex of a mammal or mammalian embryo. The presentinvention also provides for such processing, and in some preferredembodiments, additional features of preservation heretoforetraditionally thought not to be feasible commercially, or even possible,and heretofore providing a solution to those previously identified butunaddressed issues.

Obtaining Sperm

Semen, and in particular sperm cells, may be obtained and otherwiseheretofore processed from mammals in accordance with the presentinvention such as equids, bovids, felids, ovids, canids, buffalo, oxen,elk, or porcine, or other mammal species. Further, some embodiments mayprovide obtaining and processing semen or sperm cells from prized mammalspecies, endangered mammal species, rare individuals of a mammalspecies, and even from zoological specimens or individuals. Theresulting mammal or mammalian embryo may be produced in accordance withthe techniques as previously described and as further described below.Sperm samples may be established, in some embodiments, as previouslydescribed.

Sample Preservation

Sperm samples are cryopreserved, such as by freezing, using variouspreservation techniques, such as freezing in a Hepes-bufferedcrydiluent. Sperm cells may be provided as pellets or straws and may bethawed using various thawing techniques, for example, with ramspermatozoa. Other sperm samples may be provided throughout theprocessing of sperm cells, and may or may not be cryopreserved whenestablished as a sperm sample or when provided to inseminate orfertilize an egg.

One or more additives, singularly or in combination, may be introducedinto the semen, sperm cells, or sperm sample. In one embodiment, acryodiluent may be introduced into the sperm sample to preserve thesperm cells. The introduction of the additive or additives, such as acryodiluent, into the sperm sample, and in some embodiments with as acryopreservation step, potentially referred to as freezing, may maintainor enhance sperm cells, and may further maintain or enhance spermquality, sperm cell quality, such as sperm cell viability, motility,functionality, stimulation, and fertility rates, and potentially one ormore sperm cell characteristics. In other embodiments of the presentinvention, an additive or additives, such as cryodiluent, singularly orin combination, could be removed from the sperm sample. The removal ofthe cryodiluent or other additives from the sperm sample, and in someembodiments with cryopreservation steps, may maintain or enhance spermcells, and may further maintain or enhance sperm quality, such asmaintaining or enhancing sperm cell viability, motility, functionality,fertility rates, and potentially one or more sperm cell characteristics.

Sperm Processing

Sperm cells may also be maintained or enhanced in some embodiments ofthe present invention as part of sperm processing techniques, and spermquality may further be maintained or enhanced, such as maintaining orenhancing sperm cell viability, motility, functionality, and potentiallyone or more sperm cell characteristics. Accordingly, in someembodiments, the sperm sample may be preserved, as previously described,such as through cryopreservation, such as freezing, or otherpreservation techniques such as those disclosed in the previouslymentioned patent applications and the references listed in the List ofReferences to be Incorporated by Reference.

Accordingly, cryodiluent or other additives, singularly or incombination, may be introduced into the sperm sample to preserve orstimulate the sperm. In one preferred embodiment, the introduction ofthe cryodiluent or other additives into the sperm sample contributes tothe preservation of the sperm through cryopreservation, freezing thesperm sample, and therefore maintaining or enhancing the sperm cells,and further maintaining or enhancing sperm quality, such as maintainingor enhancing sperm cell viability, motility, functionality, andpotentially one or more sperm cell characteristics. As previouslymentioned, the cyrodiluent or other additives may be introduced into thesperm sample prior to sample preservation or as sample preservation. Thesample may then be processed such as through collection, handling,separation, storage, transportation, usage, fertilization, orinsemination techniques as disclosed in the previously mentioned patentapplications and the references listed in the List of References to beIncorporated by Reference.

In one embodiment, cryodiluent or other additives, singularly or incombination, may be introduced into a sperm sample previously collectedand the sample cryopreserved, such as through freezing, and followed bya thaw of the sample and subsequent processing, including collection,handling, separation, storage, transportation, usage, fertilization, orinsemination processing techniques. One such processing step may beprovided as sorting, and in some embodiments as the separation of Xchromosome bearing sperm cells from Y chromosome bearing sperm cells,potentially into a high purity population sample or samples. Variousbenefits may be achieved through such a sperm processing technique. Forexample, various methods of sorting as described in the previouslymentioned patent applications achieve a separation of X chromosomebearing sperm cells from Y chromosome bearing sperm cells whileminimizing damage to the viable sperm cells. Further, non-viable sperm,contamination, or crydiluent or other additives, for example, may beeliminated through such separation techniques. Additionally, otheraspects of sperm quality may be maintained or enhanced, particularlythat of sperm cell motility and functionality. The sperm sample mayfurther be stimulated during the processing to maintain or enhance spermquality as previously described. One such method of sperm processingknown in the art is described in U.S. Pat. No. 5,135,759, herebyincorporated by reference in this description, disclosing a flowcytometer sorting technique.

The processing of sperm cells may be performed by sorting the spermcells as previously described, or in some embodiments, accordance withthe following process. Sorting may comprise, in some embodiments, as aselecting of sperm cells based upon at least one desired characteristic,potentially sperm cell quality characteristics, such as viability,motility, functionality, stimulation, and preservation, or one or acombination of various sperm cell characteristics such as biological,chemical, physical, physiological, or functional attributes of one ormore sperm cells, such as chromosome bearing attributes of the cell. Thesperm cells may be stained, preferably with Hoechst 33342 or like stainor dye, or in combination with such stains or dyes, and the sperms cellsdifferentiated based upon the staining and selection. The cells may thenbe separated based upon the differentiation and collected. The spermcells may be so processed in accordance with the various techniques ofthose references cited herein and expressly incorporated by reference.

Example—Preserving and Processing Sample

In one embodiment of a preserving and processing technique, 200 μl ofthawed spermatozoa was placed onto a 2 ml separation gradient (90%:45%)of PURESPERM, an isotonic salt solution containing colloidal-silicaparticles coated with saline, and a Tris based diluent. The gradientpreparations were then centrifuged at 1000 g for 15 minutes. The postPURESPERM pellet was removed, slowly diluted 1:4 with warm Tris baseddiluent and centrifuged at 650 g for 3 minutes. The supernatant wasremoved and the sperm stained, incubated and sorted as previouslydescribed. The Tris based diluent was used as the staining medium andANDROHEP (Minitub, Germany) plus 20% egg yolk was used as the collectionmedium.

The above example is one of various embodiments of the inventivetechnique, providing preservation of sperm, such as throughcryopreservation or freezing, thawing the sperm, identifying the sperm,such as through staining, and sorting the sperm, such as into X and Ychromosome bearing populations.

Although the previous example provides a sperm preservation techniqueand processing technique of sperm collection, preservation, thaw andseparation, other techniques are encompassed by and explicitly disclosedin the present invention. For example, one or a combination of variouscollection, handling, separation, storage, transportation, usage,fertilization, or insemination techniques may be performed as part ofthis present inventive technique. In one embodiment, sperm may becollected, followed by separation of X chromosome bearing sperm cellsfrom Y chromosome bearing sperm cells. A preservation step prior tosperm cell separation may not be needed, for example, duringcircumstances in which the sorter apparatus is readily available aftersperm collection. The sorted sperm sample or samples may then bepreserved as an additional step and as previously described, potentiallyfor further handling, separation, storage, transportation, usage,fertilization or insemination. The sorted sperm sample may further bestimulated during the processing to maintain or enhance sperm quality aspreviously described. The next example describes one example of aprocessing technique.

Example—Processing Samples

Sorted samples were centrifuged at 700 g for 6 min. at room temperature(24 C). The supernatant was removed and the sorted sperm could be used“fresh” for AI or in an IVF system; or the remaining pellet was extended1:4 with the Hepes based cryodiluent, potentially the same diluent usedfor an original cryopreservation of the sperm, and frozen using varioustechniques, such as those previously described and as described below.The re-frozen and sorted sperm were thawed using methods such as a glasstube shaken in a 37° C. waterbath, and then could be used in an AI orIVF system.

Example—Processing Samples

The use of spermatozoa sorted from frozen-thawed pellets in the ovinemammal regarding in vitro fertilization systems. Frozen-sorted andfrozen-sorted-frozen sperm used in an ovine IVF system were slowlydiluted with 0.5 ml of IVF media, and in some embodiments, using ovineIVF protocol, and centrifuged in a Falcon tube with a tight lid at 300 gfor 6 minutes. The supernatant was removed and the remaining spermquickly placed in the IVF well at a concentration of one million motilesperm/ml. Preferably, a high standard of media preparation (i.e. use ofeggs less than 24 hours old, ultracentrifugation of egg yolk diluentsand meticulous filtering) and handling of the samples (i.e. constanttemperature) is required.

Other preservation, processing, fertilization, and inseminationtechniques need not include a separation step. For example, the spermmay be collected, followed by a preservation step and subsequenthandling, storage, transportation, usage, fertilization, orinsemination.

Furthermore, one or more preservation steps may be conducted as part ofpreservation, processing, fertilization, and insemination techniques, orcombinations thereof, such preservation in some embodiments comprisingcryopreservation, such as through freezing of the sperm sample, andsubsequent steps of thawing, occurring before, concurrent with, or afterone or more other processing techniques.

Example—Sex-Sorting and Re-Cryopreservation of Frozen-Thawed Ram Spermfor In Vitro Embryo Production

Application of sperm sorting to breeding of livestock and wildlife maybe limited when the sorter is a long distance from the male(s), aspreviously described, but would be facilitated by the sorting ofcryopreserved and thawed sperm, such as frozen-thawed sperm (Lu K H etal., Theriogenology 1999:52:1393-1405) and cryopreserving or re-freezingit. High purity sorting with maintained quality of frozen-thawed ramsperm may be achieved after processing to remove the cryodiluent. Theaim of this study was to evaluate the functional capacity offrozen-thawed sperm after sorting and a second cryopreservation/thawingstep. Frozen semen from 2 rams (n=2 ejaculates per ram) was usedthroughout. Post-thaw sperm treatments comprised (i) unsorted (Control);(ii) sorted (Frozen-Sort) and (iii) sorted then re-frozen(Frozen-Sort-Frozen). X and Y sperm were separated using a high-speedsorter (SX MoFlo(R), Cytomation, Colo., USA) after incubation withHoechst 33342 and food dye to eliminate non-viable sperm. Reanalysisrevealed high levels of purity for X- and Y-enriched samples for alltreatments (87.0+/−4.5%). For IVF, 472 IVM oocytes were inseminated with1×10(6) motile sperm/mL. After 3 h in SOF medium, oocytes weretransferred to Sydney IVF cleavage medium (Cook(R), QLD, Australia) for4 d followed by Sydney IVF blastocyst medium (Cook(R)) for an additional3 d culture in 5% O2: 5%CO2: 90% N2. Oocytes were assessed for cleavageat 24 and 48 h post-insemination (p.i.). At 52 h p.i., uncleaved oocyteswere stained with orcein for assessment of maturation and fertilization.Data from 3 replicates were analyzed by ANOVA, Chi-square and FisherExact Test. At insemination, % motile sperm (+/−SEM) was higher(P<0.001) for Frozen-Sort (85.8+/−2.4%) and Frozen-Sort-Frozen(66.7+/−7.7%) than Control (36.7+/−2.1%). Maturation rate was 95.6%(451/472). Cleavage of oocytes in a parthenogenetic control group (nosperm) was low (2/56; 3.6%). Polysperminc fertilization was low (9/451;2.0%) and did not differ among treatments.

TABLE 1 Fertilization & early embryo development of oocytes afterincubation with frozen- thawed unsorted (Control), frozen-thawed &sorted (Froz-Sort) & frozen-thawed, sorted then frozen-thawed(Froz-Sort-Froz) ram sperm. Values in parentheses are percentages. No.of No. of mature oocytes mature undergoing cleavage No. of cleavedoocytes oocytes after insemination forming blastocysts Treatmentfertilized^(d) 24 h 48 h Day 5 Day 6 Day 7 Control 40 (67.8) 26 (44.1)36 (61.0)  4 (11.1) 13 (36.1) 16 (44.4)^(a) Froz-Sort 110 (63.6)  67(38.7) 109 (63.0)  24 (22.1) 34 (31.2) 57 (52.3)^(ab) Froz-Sort-Froz 94(57.7) 71 (43.6) 91 (55.8) 23 (25.3) 347 (40.7)  59 (64.8)^(bc)^(d)Monospermic fertilization. Within column, values with differentsuperscripts differ (P < 0.05).

Fertilization and cleavage rates were consistently high acrosstreatments. Blastocyst development rate was higher for oocytesfertilized with Froz-Sort-Froz than with Control sperm. These resultsdemonstrate that frozen-thawed ram sperm can be sex-sorted for eitherimmediate or future use in an IVF system after re-cryopreservation.

The above example is one of various embodiments of the inventivetechnique, providing preservation of sperm, such as throughcryopreservation, such as freezing, thawing the sperm, sorting thesperm, such as into X and Y chromosome bearing populations, preservingthe sorted sample, such as through cryopreservation or freezing, andfurther including thawing the sperm sample for use, such as forfertilization or insemination.

Consideration should be given to fertility rates respective ofpreservation and processing procedures, such as separation forsex-sorting and cryopreservation of sperm samples, as the next Exampledescribes, and in combination with cryopreservation, thawing,processing, and subsequent cryopreservation, as disclosed in thisdescription.

Example—Effect of Dose of Sperm Processed for Sex-Sorting andCrypreservation on Fertility in Ewes

Lambs have been produced after artificial insemination (AI) with lownumbers (2-4×10⁶) of cryopreserved sex-sorted sperm. Fewer ewes werepregnant after AI with X- or Y-sorted frozen-thawed (25%, 15%respectively) than with a commercial dose of unsorted frozen-thawedsperm (54%). The object of the present study was to determine theminimum numbers of sorted frozen-thawed sperm required to obtainpregnancy rates similar to those obtained with unsorted sperm.

A sample of sperm from single ejaculates of 2 rams was stained,incubated, analyzed and sorted using a modified high speed cell sorter(MoFlo®, Cytomation, Fort Collins, Colo., USA) as previously described.Sperm were processed at 15,000-18,000/serc without sex-sorting into 10ml centrifuge tubes pre-soaked with 1% BSA in sheath fluid containing0.2 ml Tris-buffered medium and 20% egg yolk (v/v). For every sample,1.3×10⁶ sperm were sex-sorted and analyzed to determine purity. Sortedand unsorted (control) samples were extended with a zwitterions-buffereddiluent containing 13.5% egg yolk and 6% glycerol and frozen as 250 ulpellets containing 5×10⁶ sperm. The time of estrus was controlled in 144Merino ewes by progestagen sponges (FGA, Vetrepharm A/Asia, Sydney)inserted intravaginally for 12 days and an injection of 400 I.U of PMSG(Pregnecol, Vetrepharm A/Asia) at sponge removal (SR). Thirty-six hafter SR 134 ewes were injected with 40 μg GnRH (Fertagyl®, Intervet) tocontrol the time of ovulation. One hundred and eleven ewes wereinseminated in the uterus by laparoscopy 57-60 h after SR with 5, 10, 20or 40×10⁶ sorted or unsorted frozen-thawed sperm. Thirteen ewes notgiven GnRH were inseminated with a commercial dose of unsortedfrozen-thawed sperm. Thirteen ewes not given GnRH were inseminated witha commercial dose of unsorted frozen-thawed sperm 57-58 h after SR.Pregnancy was diagnosed by ultrasound on d53. The data were analyzed byChi-square.

Sperm motility after thawing was 37.8+/−1.78% (sorted) and 42.9+/−0.93%(unsorted). Seven of 13 (53.8%) ewes not given GnRH were pregnant. Ofthe GnRH-treated ewes the proportion pregnant was affected by the numberof sperm inseminated (p<0.05) but not by ram or type of sperm (p>0.05).For ewes inseminated with sorted or unsorted (control) frozen-thawedsperm, pregnancy rate was higher for inseminates of 10 and 40×10⁶ thanfor 5 and 20×10⁶ sperm (Table 1). The results suggest that a minimum of40×10⁶ sorted frozen-thawed sperm inseminated close to the time ofovulation are required to obtain commercially acceptable pregnancyrates.

TABLE 1 Pregnancy after intrauterine insemination of ewes withfrozen-thawed control and sorted ram sperm. Dose No. ewes No. ewes %ewes (×10⁶ sperm) inseminated pregnant pregnant 5 30 10 33.3^(a) 10 2816 57.1^(b) 20 29 10 34.5^(a) 40 23 16 69.6^(a) ^(ab)Within columnsdifferent superscripts differ (p < 0.05). ^(a)This research wassupported by XY, Inc; CO, USA; The Australian Research Council,Vetrephann A/Asia.

The invention can further include a sperm sample, such as a straw, andin some embodiments straws utilized in IVF, produced in accordance withany of the above described embodiments of the invention, such as any ofthe processing, stimulation, preservation, fertilization, andinsemination techniques, and be of maintained or enhanced sperm quality,such as a straw of desired viability, motility, functionality, or othercharacteristics, or combinations of characteristics, potentiallyresulting in desirable levels of fertility rates, and in someembodiments, particularly for equine mammals. The sperm sample or strawmay be particularly suited for individual production embryos.

The invention can further include a mammal produced in accordance withany of the above described embodiments of the invention, or can includea mammal of predetermined sex in accordance with the various embodimentsof the invention that provide sperm cell insemination samples having anenriched population of either X-chromosome bearing sperm cells orenriched population of Y-chromosome bearing sperm cells, or a mammalproduced in accordance with any embodiment of the invention in which asperm cell insemination sample containing a low number of sperm cellscompared to the typical number used to inseminate that particularspecies of mammal is used, or elk progeny produced in accordance withthe invention as described above.

The invention further includes various processing, preservation,stimulation, fertilization, and insemination techniques as disclosedherein and as disclosed in the previously mentioned patent applicationsand references. Accordingly, the various semen and sperm cell processingsystems and systems of preservation, stimulation, fertilization, andinsemination, embodiments, in various embodiments addressing spermquality such as one or more sperm cell characteristics, such asviability, motility, functionality, or fertilization rates consistentwith the disclosures of the previously mentioned patent applications andreferences. Further, sperm cell characteristics may be addressed withinthe context of various collection, handling, separation, storage,transportation, usage, fertilization, or insemination techniques, and inthose or other various embodiments, within the context of assaying,testing, or determining the biological, chemical, physical,physiological, or functional attributes of sperm cells. Therefore,systems of the present invention may provide sperm cell processing,stimulation, preservation, fertilization, and insemination, for example,incorporating flow sorting techniques, high purity separationtechniques, low dose fertilization and insemination techniques,heterospermic insemination procedures, such as to assess comparativeviability, motility, function, or fertility processed in variouspressure environments within a sorter, as but a few examples.

The disclosure incorporated by reference, such as the various examplesprovided of separating X chromosome bearing sperm cells from Ychromosome bearing sperm cells, and other disclosed techniques ofcollection, handling, separation, storage, transportation, usage,fertilization, and insemination are not meant to limit the presentinvention to any particular embodiment, whether apparatus, method, orotherwise. The descriptions incorporated by reference and the variousexamples should not be construed to limit the present invention to onlytechniques for sperm sorting or only techniques for sperm preservation.This disclosure, however, may be understood to incorporate the varioustechniques in the context of the various embodiments of the presentinvention. Further, the present invention should be considered toincorporate such techniques of sperm processing, preservation,stimulation, fertilization, and insemination consistent with thefeatures disclosed.

As can be easily understood from the foregoing, the basic concepts ofthe present invention may be embodied in a variety of ways. It involvesboth a sperm cell process system including both techniques as well asdevices to accomplish sperm cell processing. In this application,various sperm cell processing techniques are disclosed as part of theresults shown to be achieved by the various devices described and assteps which are inherent to utilization. They are simply the naturalresult of utilizing the devices as intended and described. In addition,while some devices are disclosed, it should be understood that these notonly accomplish certain methods but also can be varied in a number ofways. Importantly, as to all of the foregoing, all of these facetsshould be understood as encompassed by this disclosure.

Further, each of the various elements of the invention and claims mayalso be achieved in a variety of manners. This disclosure should beunderstood to encompass each such variation, be it a variation of anembodiment of any apparatus embodiment, a method or process embodiment,or even merely a variation of any element of these. Particularly, itshould be understood that as the disclosure relates to elements of theinvention, the words for each element may be expressed by equivalentapparatus terms or method terms—even if only the function or result isthe same. Such equivalent, broader, or even more generic terms should beconsidered as encompassed in the description of each element or action.Such terms can be substituted where desired to make explicit theimplicitly broad coverage to which this invention is entitled. As butone example, it should be understood that all actions may be expressedas a means for taking that action or as an element which causes thataction. Similarly, each physical element disclosed should be understoodto encompass a disclosure of the action which that physical elementfacilitates. Regarding this last aspect, as but one example, thedisclosure of a “cryopreserver” should be understood to encompassdisclosure of the act of “cryopreserving”—whether explicitly discussedor not—and, conversely, were there effectively disclosure of the act of“cryopreserving”, such a disclosure should be understood to encompassdisclosure of a “cryopreserver” and even a “means for cryopreserving.”Such changes and alternative terms are to be understood to be explicitlyincluded in the description.

Any acts of law, statutes, regulations, or rules mentioned in thisapplication for patent; or patents, publications, or other referencesmentioned in this application for patent are hereby incorporated byreference. In addition, as to each term used it should be understoodthat unless its utilization in this application is inconsistent withsuch interpretation, common dictionary definitions should be understoodas incorporated for each term and all definitions, alternative terms,and synonyms such as contained in the Random House Webster's UnabridgedDictionary, second edition are hereby incorporated by reference.Finally, all references listed in the information statement filed withthe application are hereby appended and hereby incorporated byreference, however, as to each of the above, to the extent that suchinformation or statements incorporated by reference might be consideredinconsistent with the patenting of this/these invention(s) suchstatements are expressly not to be considered as made by theapplicant(s).

Thus, the applicant(s) should be understood to claim at least: i) eachof the sperm cell processing methods as herein disclosed and described,ii) the related systems, devices, and multiple apparatus disclosed anddescribed, iii) similar, equivalent, and even implicit variations ofeach of these systems and methods, iv) those alternative designs whichaccomplish each of the functions shown as are disclosed and described,v) those alternative designs and methods which accomplish each of thefunctions shown as are implicit to accomplish that which is disclosedand described, vi) each feature, component, and step shown as separateand independent inventions, vii) the applications enhanced by thevarious systems or components disclosed, viii) the resulting productsproduced by such systems or components, and ix) methods and apparatusessubstantially as described hereinbefore and with reference to any of theaccompanying examples, x) the various combinations and permutations ofeach of the elements disclosed, and xi) each potentially dependent claimor concept as a dependency on each and every one of the independentclaims or concepts presented.

The Applicant may have presented claims with an set of initialdependencies. Support should be understood to exist to the degreerequired under new matter laws—including but not limited to EuropeanPatent Convention Article 123(2) and United States Patent Law 35 USC 132or other such laws—to permit the addition of any of the variousdependencies or other elements presented under one independent claim orconcept as dependencies or elements under any other independent claim orconcept.

Further, if or when used, the use of the transitional phrase“comprising” is used to maintain the “open-end” claims herein, accordingto traditional claim interpretation. Thus, unless the context requiresotherwise, it should be understood that the term “comprise” orvariations such as “comprises” or “comprising”, are intended to implythe inclusion of a stated element or step or group of elements or stepsbut not the exclusion of any other element or step or group of elementsor steps. Such terms should be interpreted in their most expansive formso as to afford the applicant the broadest coverage legally permissible.

What is claimed is:
 1. A method of processing sperm cells, comprising:obtaining once-cryopreserved sperm cells of a nonhuman mammal; thawingsaid once-cryopreserved sperm cells; sorting said once-cryopreservedsperm cells subsequent to thawing to yield an X-chromosome bearing spermcell population or a Y-chromosome chromosome bearing sperm cellpopulation; and cryopreserving said X-chromosome bearing sperm cellpopulation or said Y-chromosome bearing sperm cell population to yieldtwice-cryopreserved sorted sperm cell populations effective upon thawingof fertilizing an egg which develops into a blastocyst of said nonhumanmammal.
 2. The method of claim 1, further comprising staining saidonce-cryopreserved sperm cells prior to sorting said once-cryopreservedsperm cells.
 3. The method of claim 2, further comprising flow sortingsaid once-cryopreserved sperm cells subsequent to staining with a flowcytometer.
 4. The method of claim 3, further comprising thawing saidX-chromosome bearing population or said Y-chromosome bearing populationof twice-cryopreserved sperm cells.
 5. The method of claim 4, furthercomprising fertilizing said egg of said non-human mammal with saidX-chromosome bearing population or said Y-chromosome bearing populationof twice-cryopreserved sperm cells.
 6. The method of claim 5, whereinsaid egg of said non-human mammal fertilized with said X-chromosomebearing population or said Y-chromosome bearing population oftwice-cryopreserved sperm cells develops into said blastocyst.
 7. Themethod of claim 3, further comprising containing said X-chromosomebearing population or said Y-chromosome bearing population oftwice-cryopreserved sperm cells within an artificial insemination strawprior to said cryopreserving.
 8. The method of claim 1, furthercomprising fertilizing said egg of said nonhuman mammal with said spermsample by intracervical or intrauterine placement of said X-chromosomebearing population or said Y-chromosome bearing population oftwice-cryopreserved sperm cells.
 9. The method of claim 8, wherein saidX-chromosome bearing population or said Y-chromosome bearing populationof twice-cryopreserved sperm cells upon thawing contains an increasedpercentage of viable sperm cells as compared to said once-cryopreservedsperm cells upon thawing.
 10. The method of claim 8, wherein saidX-chromosome bearing population or said Y-chromosome bearing populationof twice-cryopreserved sperm cells upon thawing contains a greaterpercentage of motile sperm cells as compared to said once cryopreservedsperm cells upon thawing.
 11. The method of claim 5, further comprisingfertilizing said egg of said nonhuman mammal with said X-chromosomebearing population or said Y-chromosome bearing population oftwice-cryopreserved sperm cells by in-vivo fertilization.
 12. The methodof claim 5, wherein fertilizing said egg of said nonhuman mammal eggwith said X-chromosome bearing population or said Y-chromosome bearingpopulation of twice-cryopreserved sperm cells increases blastocystdevelopment rate compared to said thawed once-cryopreserved sperm cells.13. The method of claim 10, wherein said X-chromosome bearing populationor said Y-chromosome bearing population of twice-cryopreserved spermcells contains at least 83 percent motile sperm cells.
 14. The method ofclaim 4, further comprising artificially inseminating a female of saidnonhuman mammal by intracervical or intrauterine placement of saidX-chromosome bearing population or said Y-chromosome bearing populationof twice-cryopreserved sperm cells.
 15. The method of claim 14, whereinartificial insemination of said female of said nonhuman mammal byintracervical or intrauterine placement of said X-chromosome bearingpopulation or said Y-chromosome bearing population oftwice-cryopreserved sperm cells increases percentage pregnancy rate ascompared to said once-cryopreserved sperm cells.
 16. The method of claim4, further comprising artificially inseminating a female of saidnonhuman mammal by intracervical or intrauterine placement of saidX-chromosome bearing population or said Y-chromosome bearing populationof twice-cryopreserved sperm cells in an unthawed condition.
 17. Themethod of claim 16, further comprising: superovulating said female ofsaid nonhuman mammal; and producing at least one embryo by artificiallyinseminating said female of said nonhuman mammal by intracervical orintrauterine placement of said X-chromosome bearing population or saidY-chromosome bearing population of twice-cryopreserved sperm cells. 18.The method of claim 17, further comprising the step of transferring saidat least one embryo to a recipient female.
 19. The method of claim 1,wherein said once-cryopreserved sperm cells and said twice-cryopreservedsperm cells are frozen in a cryodiluent.
 20. The method of claim 19,wherein said cryodiluent includes a zwitterionic buffer.
 21. The methodof claim 20, wherein said zwitterionic buffer comprises HEPES buffer.22. The method of claim 21, wherein said cryodiluent includes an amountof glycerol.
 23. The method of claim 22, wherein said amount of glycerolcomprises 6% by volume of said cryodiluent.
 24. The method of claim 19,wherein said cryodiluent further comprising an amount of egg yolk. 25.The method of claim 24, wherein said amount of egg-yolk comprises about13.5% egg yolk.
 26. The method of claim 19, further comprising isolatingsaid thawed sperm cells by a particle density gradient prior to saidsorting of said thawed sperm cells.
 27. The method of claim 26, whereinthawing said cryopreserved sperm cells occurs at about 37° C.
 28. Amethod of processing sperm cells, comprising: obtainingonce-cryopreserved sperm cells of a non-human mammal; isolating saidonce-cryopreserved sperm cells by particle density gradientcentrifugation at about 37° C., wherein said particle density gradientcentrifugation includes a density gradient 90%:45% of an isotonic saltsolution containing silane-coated silica particles and a Tris baseddiluent; sorting isolated once-cryopreserved sperm cells based onbearing either an X chromosome or a Y chromosome; and cryopreservingsaid once-cryopreserved sperm cells to yield twice-cryopreserved spermcells sorted into an X-chromosome bearing population or a Y-chromosomebearing population effective upon thawing of fertilizing an egg whichdevelops into a blastocyst of said non-human mammal, said cryodiluentincluding a zwitterionic HEPES buffer, 6% glycerol by volume, and 13.5%egg yolk.